Packing-induced conformational and functional changes in the subunits of α-crystallin

Abstract

The heteroaggregate α-crystallin and homoaggregates of its subunits, αA- and αB-crystallins, function like molecular chaperones and prevent the aggregation of several proteins. Although modulation of the chaperone-like activity of α-crystallin by both temperature and chaotropic agents has been demonstrated in vitro, the mechanism(s) of its regulationin vivo have not been elucidated. The subunits of α-crystallin exchange freely, resulting in its dynamic and variable quaternary structure. Mixed aggregates of the α-crystallins and other mammalian small heat shock proteins (sHSPs) have also been observedin vivo. We have investigated the time-dependent structural and functional changes during the course of heteroaggregate formation by the exchange of subunits between homoaggregates of αA- and αB-crystallins. Native isoelectric focusing was used to follow the time course of subunit exchange. Circular dichroism revealed large tertiary structural alterations in the subunits upon subunit exchange and packing into heteroaggregates, indicating specific homologous and heterologous interactions between the subunits. Subunit exchange also resulted in quaternary structural changes as demonstrated by gel filtration chromatography. Interestingly, we found time-dependent changes in chaperone-like activity against the dithiothreitol-induced aggregation of insulin, which correlated with subunit exchange and the resulting tertiary and quaternary structural changes. Heteroaggregates of varying subunit composition, as observed during eye lens epithelial cell differentiation, generated by subunit exchange displayed differential chaperone-like activity. It was possible to alter chaperone-like activity of preexisting oligomeric sHSPs by alteration of subunit composition by subunit exchange. Our results demonstrate that subunit exchange and the resulting structural and functional changes observed could constitute a mechanism of regulation of chaperone-like activity of α-crystallin (and possibly other mammalian sHSPs) in vivo.